In the industrial production of monoclonal antibodies and Fc fusion proteins, purification is frequently carried out using chromatography. Protein A from Staphylococcus aureus has long been used as affinity ligand in such applications, due to the native affinity of Protein A for the Fc portion of IgG. Protein A in its entirety, as well as the five individual Fc-binding domains thereof, have subsequently served as starting points for the rational design of engineered affinity ligands with improved properties.
For example, the B domain of Protein A served as a starting point for the creation of the engineered, IgG Fc-binding, single-domain protein Z (Nilsson B et al, Protein Eng 1(2):107-13, 1987). In order to improve the stability of this protein in alkaline conditions, such as those employed in cleaning-in-place procedures during industrial chromatographic processes, Linhult et al (Proteins 55(2):407-16, 2004) proposed a variant of protein Z comprising mutated asparagine residues. This variant was further developed by GE Healthcare, Uppsala, Sweden, into the commercial product MAbSelect™ SuRe. Additional Z variants with IgG Fc affinity are disclosed in WO2009/146755.
Mutated binding proteins based on protein Z, in which the native IgG Fc binding has been modified, are described in WO2009/080811.
Native Protein A, with its five individual IgG Fc-binding domains, has been shown to bind only 2 molecules of IgG, or 3 molecules of recombinant Fc-fragment, despite the fact that each of the five domains has the capacity to bind one IgG (Birger Jansson, PhD Thesis, Stockholm, Sweden, 1996, ISBN 91-7170-656-9).
Despite the comparable success of currently used IgG Fc affinity ligands, there is a continued need for improvement, especially with regard to the combined requirements of improving stoichiometry and at the same time maintaining, and ideally improving, stability towards acidic and alkaline conditions. The continued provision of agents with affinity for IgG Fc remains a matter of substantial interest.